Device for performing a diagnostic test and methods for use thereof

ABSTRACT

Assay cassettes and testing devices that can be used to provide rapid, accurate, affordable, laboratory-quality testing at the point of care. Such assay cassettes and testing devices are designed to provide rapid, quantitative test results in a point-of-care setting or the like. Likewise, such assay cassettes and testing devices may eliminate or replace expensive, centralized clinical testing equipment and technical personnel. Such testing device may include automated data reporting and decision support. Methods for performing point of care diagnostic tests are also disclosed.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Prov. Pat.App. Ser. No. 61/625,368 filed 17 Apr. 2012 and U.S. Prov. Pat. App.Ser. No. 61/740,975 filed 21 Dec. 2012, the entireties of which areincorporated herein by reference.

BACKGROUND

Sampling and testing of biological samples and body fluids (e.g.,saliva, blood, urine, fecal matter, foods, plants, fish, minerals,animals, etc.) is common for both testing and monitoring humans, fish,animals, and plants for any number of biochemical or physiologicalconditions and, of course, for determining the general state of healthof an organism. For example, sampling and testing of human body fluidsis often performed for point-of-care testing (“POCT”). POCT is definedas medical testing at or near the site of patient care. The drivingnotion behind POCT is to perform and provide the test conveniently andimmediately to the patient. This increases the likelihood that thepatient, physician, and care team will receive the results more quicklyand allows for immediate clinical management decisions to be made. POCTexamples include, but are not limited to, blood glucose testing,metabolic testing (e.g., thyroid stimulating hormone), blood gas andelectrolytes analysis, rapid coagulation testing, rapid cardiac markersdiagnostics, drugs of abuse screening, urine testing, pregnancy testing,fecal occult blood analysis, food pathogen screening, hemoglobindiagnostics, infectious disease testing, cholesterol screening, cancertesting (e.g. PSA), hormone testing (hCG, LH, FSH), cardiac (troponin),pulmonary, gastroenterology (e.g., H. pylori antibodies), urology,dermatology, neurology, pediatrics, surgical, and public health (Ebola,cholera, HIV), testing and combinations thereof.

One testing method that is often employed for POCT and more conventionaltesting involves the use of lateral-flow chromatographic immunoassaycassettes. Lateral-flow chromatographic immunoassay cassettes can beused to easily and quickly obtain a variety of qualitative resultsrelating to a number of biochemical and physiological conditions anddisease states of an individual. These kinds of tests require the enduser to simply add a sample to the cassette and then observe the resulta few minutes later. Since such rapid and easy-to-use tests are userfriendly, they are very popular in both the professional and consumermarkets nowadays. Such tests are also widely used in areas where accessto trained health care professionals is limited or where access toproper medical facilities is limited (e.g., poor areas, developingcountries, war zones, etc.).

Lateral flow chromatographic immunoassay methods and devices have beendescribed extensively. See, e.g., Gordon and Pugh, U.S. Pat. No.4,956,302; H. Buck, et al., WO 90/06511; T. Wang, U.S. Pat. No.6,764,825; W. Brown, et al., U.S. Pat. No. 5,008,080; Kuo and Meritt,U.S. Pat. No. 6,183,972, EP 00987551A3. Such assays involve thedetection and determination of an analyte substance that is a member ofa specific binding pair consisting of a ligand and a receptor. Theligand and the receptor are related in that the receptor specificallybinds to the ligand, being capable of distinguishing a specific ligandor ligands from other sample constituents having similarcharacteristics. Immunological assays involving reactions betweenantibodies and antigens are one such example of a specific bindingassay. Other examples include DNA and RNA hybridization reactions andbinding reactions involving hormones and other biological receptors. Onewell-known commercial embodiment of this technique is the ClearblueOne-Step Pregnancy Test.

Lateral flow chromatographic immunoassay test cassettes have a number ofdesirable characteristics including their ease of use and broadapplicability to a variety of analytes. Likewise, immunoassay procedurescapable of being carried out on a test strip and which can beadministered in the field or other locations where medical testinglaboratories are not readily available have provided a great benefit tothe diagnosis and control of disease. Currently, however, such lateralflow chromatographic immunoassay tests are generally only capable ofproviding qualitative results. That is, while currently availablelateral flow chromatographic immunoassay test cassettes and cassettereader apparatuses are particularly well-suited for telling apractitioner whether or not one or more test substances are present in asample above a given detection limit, they are poorly suited forproviding quantitative results. There is an ongoing need in the art fordevices and methods that combine the ease of use characteristics oflateral flow chromatographic immunoassay tests with systems that aredesigned to provide quantitative results. Such devices and methods may,for example, allow medical practitioners to diagnose, monitor, andmanage a variety of conditions at the point of care (e.g., chair-side oressentially anywhere in the world) without being tied to a medicalfacility or a testing laboratory.

BRIEF SUMMARY

Devices and methods for performing point of care diagnostic tests fordetecting and quantifying at least one analyte in a biological sample(e.g., a body fluid). Disclosed herein are assay cassettes and testingdevices that can be used to provide rapid, accurate, affordablelaboratory-quality testing at the point of care. Such assay cassettesand testing devices are designed to provide rapid, quantitative testresults in a point-of-care setting or the like where, in the past, onlyqualitative or semi-quantitative results have typically been available.Likewise, such assay cassettes and testing devices may eliminate orreplace expensive, centralized clinical testing equipment and technicalpersonnel. Such testing devices may include automated data reporting anddecision support.

In one embodiment, a diagnostic test system is disclosed. The systemincludes a lateral-flow chromatographic assay cassette and a compact,portable testing device that includes data collection and data analysiscapabilities. The testing device is configured to interface with andanalyze output of the lateral-flow chromatographic assay cassette.

In one embodiment, the lateral-flow chromatographic assay cassette mayinclude a capture ligand capable of capturing and localizing at leastone analyte of interest in a sample on an analysis surface of thelateral-flow chromatographic assay cassette, at least one reporterconfigured for interacting with at least one of the analyte of interestor the capture ligand, and at least a first calibration standard and asecond calibration standard configured to provide at least a two-pointcalibration curve.

In another embodiment, the lateral flow chromatographic assay cassettemay include a test strip and a separate calibration strip. In thisembodiment of a lateral flow chromatographic assay cassette, a testsample (i.e., a sample containing an unknown amount of an analyte ofinterest) may be run in parallel with a calibration standard (i.e., asample containing a known amount of the analyte of interest). Theresponse to the known amount of the analyte of interest in thecalibration standard on the lateral flow immunoassay device may be usedto generate a calibration curve that can be used to quantify the amountof the analyte of interest in the test sample.

The lateral flow chromatographic assay cassette that includes a teststrip and a separate calibration strip cassette may include a base, anabsorbent test strip for analyzing an analyte of interest in anexperimental sample positioned above the base, and an absorbentcalibration strip for running at least one calibration standardpositioned above the base in proximity to the absorbent test strip. Thedevice further includes a first sample application zone positionedbetween a distal end and a proximal end the first absorbent strip, and asecond sample application zone positioned between a distal end and aproximal end of the second absorbent strip. A volume of a liquid testsample applied to the first sample application zone and a volume of aliquid calibration standard applied or deposited to the second sampleapplication zone each diffuse (i.e., wick) through their respectiveabsorbent strips from the distal end to the proximal end. Accordingly,the analyte of interest, if present in the experimental sample, and thecalibration standard interact with at least a first reporter (e.g., anantibody) immobilized on the first and second absorbent strips to yielda detectable signal.

The testing device includes a testing apparatus that is configured forcollecting data from the lateral-flow chromatographic assay cassette. Inone embodiment, the testing device includes a testing apparatus that isconfigured to be physically coupled to a handheld device (e.g., asmartphone). The testing apparatus couples the lateral-flowchromatographic assay cassette to the handheld device in proximity to alight source, the light source being capable of transmitting at leastone wavelength of light configured to yield a detectable signal from thereporter(s), and a detector positioned to capture the detectable signalfrom the reporter(s). In another embodiment, the testing apparatus maybe a stand-alone device that includes its own light source, optics, datacapture capabilities, and the like. In such an embodiment, the testingapparatus may be configured to collect assay data from an assay cassetteand transfer it to a handheld device (e.g., a smartphone) for analysisand reporting.

In addition, the system described herein may include an interpretivealgorithm stored in a computer readable format and electronicallycoupled to a handheld device, wherein the interpretive algorithm isconfigured to (i) calculate a calibration curve based on at least one ofa the first calibration standard and the second calibration standard ora known amount of an analyte of interest and a blank region and then(ii) convert the detectable signal from the reporter(s) to a numericalvalue related to the presence or amount of the at least one analytepresent in a sample. The interpretive algorithm may be included in anon-board computing system of the handheld device or the interpretivealgorithm may be stored remotely in a computer storage medium that isaccessible by the handheld device.

In another embodiment, a method for detecting at least one analyte ofinterest in a sample is disclosed. The method includes (1) providing alateral-flow chromatographic assay cassette as described herein above,(2) providing a testing device as described herein above, and (3)applying a liquid sample that includes at least one analyte of interestto the lateral-flow chromatographic assay cassette. The method furtherincludes (4) inserting the lateral-flow chromatographic assay cassetteinto the testing apparatus, (5) illuminating the lateral-flowchromatographic assay cassette with the light source of the handhelddevice in order to yield a detectable signal from the reporter(s), and(6) querying the interpretive algorithm for (i) calculating thecalibration curve and then (ii) converting the detectable signal from afirst reporter to a numerical value related to the presence or amount ofthe at least one analyte present in a sample.

These and other objects and features of the present invention willbecome more fully apparent from the following description and appendedclaims, or may be learned by the practice of the invention as set forthhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only illustrated embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1 illustrates a perspective view of a diagnostic test system,according to one embodiment of the present disclosure;

FIGS. 2A and 2B illustrates a lateral flow immunoassay device accordingto one embodiment of the present invention;

FIGS. 3A and 3B illustrates a lateral flow immunoassay device accordingto another embodiment of the present invention;

FIG. 4A illustrates a plan view of a diagnostic test system thatincludes a digital camera device and a testing apparatus configured tocouple the lateral-flow chromatographic immunoassay cassette to thedigital camera device;

FIG. 4B illustrates a side view of the diagnostic test system of FIG.4A;

FIG. 5A illustrates an exploded view of the diagnostic testing systemthat is illustrated in FIGS. 4A and 4B;

FIG. 5B illustrates a view of a component of the diagnostic test systemshown in FIG. 5A, wherein the component includes a light sealingfeature;

FIG. 6 illustrates a view of a diagnostic test system that includes anindexing feature for aligning the digital camera device and the testingapparatus;

FIG. 7A is a cut-away view of a testing apparatus of a diagnostic testsystem illustrating a target device configured for normalizing and/orcalibrating the light source and the detector of the diagnostic testsystem;

FIG. 7B is a cut-away view of a testing apparatus of a diagnostic testsystem illustrating a mechanical interlock feature configured tointerlock with a corresponding second mechanical interlock feature on alateral-flow chromatographic assay cassette;

FIG. 8 illustrates a lateral-flow chromatographic assay cassettepackaging system that includes a tracking feature readable by thehandheld device;

FIG. 9 illustrates a two point calibration curve according to oneembodiment of the present disclosure; and

FIG. 10 is a decision tree schematically illustrating a decision supportalgorithm according to one embodiment of the present disclosure.

DETAILED DESCRIPTION

Devices and methods for performing point of care diagnostic tests fordetecting and quantifying at least one analyte in a biological sample(e.g., a body fluid). Disclosed herein are assay cassettes and testingdevices that can be used to provide rapid, accurate, affordablelaboratory-quality testing at the point of care. Such assay cassettesand testing devices are designed to provide rapid, quantitative testresults in a point-of-care setting or the like where, in the past, onlyqualitative or semi-quantitative results have typically been available.Likewise, such assay cassettes and testing devices may eliminate orreplace expensive, centralized clinical testing equipment and technicalpersonnel. Such testing device may include automated data reporting anddecision support.

In one embodiment, a diagnostic test system is disclosed. The systemincludes a lateral-flow chromatographic assay cassette and a testingdevice that includes data collection and data analysis capabilities. Thetesting device is configured to interface with and analyze output of thelateral-flow chromatographic assay cassette.

I. Diagnostic Test Systems

Referring to FIG. 1, perspective view of a diagnostic test system 100 isillustrated. The diagnostic test system 100 includes a lateral-flowchromatographic assay cassette 105 and means for collecting assay datafrom the lateral-flow chromatographic assay cassette 105.

The lateral-flow chromatographic assay cassette 105 includes a plastichousing 107 containing a test strip, which is generally a plastic striplaminated with porous material that permits lateral flow of liquid. Theillustrated lateral-flow chromatographic immunoassay cassette 105includes a sample application zone 110 and an analysis zone 130.

When a sample 120 is applied to the lateral-flow chromatographicimmunoassay cassette 105 at the sample application zone 110, the sample120 diffuses through the strip in flow direction 125 toward the analysiszone 130. In the embodiment illustrated in FIG. 1, the analysis zone 130includes a test line 140 that includes at least one capture ligandselected for capturing at least one analyte of interest in the sample120. The analysis zone 130 further includes at least first and secondcalibration standard lines 150 a and 150 b. Additionally, the analysiszone may include a positive control line 170 that may be configured toprovide an indication regarding whether or not sample has diffusedthough the strip and whether or not the assay is functioning. Forexample, the positive control line 170 may include a water soluble dyethat is positioned and configures to indicate that the sample has flowedthe length of/travered the test strip.

The analyte(s) of interest, the first and second calibration standards,and the positive control can be detected on their various target lines,140, 150 a, 150 b and 170, respectively, with various reporters. Thereporters 160 for each of the various target lines, 140, 150 a, 150 band 170, may be the same or different. Examples of suitable reportersinclude, but are not limited to, visible and fluorescent dyes, latexbeads, enzymes, gold nanoparticles, silver nanoparticles, titaniumnanoparticles, europium fluorophores, quantum dots, and the like.Quantum dots are nano-scale materials that can produce excited emissionat particular wavelengths depending on their size and shape. Quantumdots can be used in immunoassays where dyes have traditionally beenused. However, quantum dots are generally superior to traditionalorganic dyes on several counts: quantum dots are typically much brighterthat organic dyes (owing to their high extinction coefficients combinedwith a comparable quantum yield to fluorescent dyes) as well as theirstability (i.e., much less photobleaching). For example, it has beenestimated that quantum dots are 20 times brighter and 100 times morestable than traditional fluorescent reporters.

Emission from the various reporters can be excited by a number ofsources. In the illustrated embodiment, an LED light source 180 is usedilluminate the analysis zone 130 of the lateral flow assay cassette 105.Illumination by the light source 180 may produce a detectable signalthat includes at least one of emission (e.g., fluorescence), color,reflectance, diffuse scattering (i.e., scattering and absorbance),elastic light scattering, chemiluminescence, chemifluorescence,transmission, plasmon surface resonance, or absorbance from thereporters. A lens 190 (e.g., a collimating lens) and a detector 195(e.g., a CCD or CMOS camera) are used to collect data from the reportersand the first and second calibration standards.

When the sample 120 is applied to the diffusion strip of thelateral-flow chromatographic assay cassette 105, the liquid in thesample carries the analyte of interest through the diffusion strip inflow direction 125 into the analysis zone 130 where it can be capturedby the capture ligand line 140. The first and second calibrationstandard lines 150 a and 150 b are selected to provide a detectablesignal that correlate to non-zero concentration values of the analyte ofinterest. For example, the first and second calibration standard lines150 a and 150 b may include an amount of the analyte of interest oranother material pre-bound to the diffusion strip of the lateral-flowchromatographic assay cassette 105. The reporter 160 may be a diffusiblematerial that can bind to the capture ligand line 150 and the first andsecond calibration standards 150 a and 150 b in an amount proportionalto the amount of bound ligand is present in each line. In response toillumination by the light source, the reporter 160 bound to each oflines 140, 150 a, and 150 b provides a signal that can be used tocalculate a calibration curves and, in turn, determine the concentrationof the analyte of interest in the sample 120. A more detailed discussionof methods for deriving analyte concentration from the data of the firstand second calibration standards 150 a and 150 b and the capture line140 is discussed in greater detail elsewhere herein.

In one type of lateral-flow chromatographic immunoassay cassette, thetest strip is divided into four domains, which can be made of only onekind of material or several kinds of material (e.g., up to fourdifferent kinds of materials). The first domain is for sample addition.It functions to remove viscous and particulate materials in the sampleand also to condition the sample solution for the reactions in thefollowing domains. The second domain is a mobile-phase with a colorconjugate. In one embodiment, the color conjugate may be made fromconjugation between a visible color marker (e.g., colored beads,colloidal gold, fluorescent dyes, etc.) and a detection antibody. Thedetection antibody can bind a specific antigen in the sample (e.g., ananalyte of interest or a positive control substance) and forms anantigen-color conjugate complex. The third domain of the lateral-flowchromatographic immunoassay cassette is a solid-phase with immobilizedcapture antibody. The capture antibody can bind the antigen of theantigen-color conjugate complex and forms capture antibody-antigen-colorconjugate complex sandwich. The fourth domain is for solutionabsorption. It draws sample solution towards it continuously.

During the testing, sample added to the first domain flows to the seconddomain. If the antigen is present in the sample, it will bind the colorconjugate to form antigen-color conjugate complex. This complex thenmigrates to the third domain to bind the capture antibody and forms thecapture antibody-antigen-color conjugate complex sandwich. Since thecapture antibody is immobilized in the third domain, the sandwich showsas a visible color signal or a fluorescent signal, depending on the dyetype, on the site of the capture antibody. If there is no antigen in thesample, no sandwich can be formed and hence no visible color signal canbe seen in the third domain. This is a so-called non-competitiveimmunoassay or a sandwich assay where the amount of signal is directlyproportional to the concentration of the analyte of interest in thesample.

Lateral-flow chromatographic immunoassay cassettes can also be adaptedfor competitive immunoassays. In a competitive immunoassay, the analyteof interest in the unknown sample competes for binding to an antibodywith a labeled analyte. In a competitive assay, the labeled analyte isable to provide a known signal. In the assay, the amount of labeledanalyte bound to the antibody is measured and any reduction in the knownsignal is attributed to the presence of the analyte in the sample. Thatis, in this method, the response will be inversely related to theconcentration of analyte in the unknown. This is because the greater theresponse, the less antigen in the unknown was available to compete withthe labeled antigen.

Lateral-flow chromatographic immunoassay cassettes may be adapted forassaying a number of different analyte types. For example, immunoassaycassettes have been adapted or may in the future be adapted for bloodglucose testing, metabolic testing (e.g., thyroid stimulating hormone),blood gas and electrolytes analysis, rapid coagulation testing, rapidcardiac markers diagnostics, drugs of abuse screening, urine testing,pregnancy testing, fecal occult blood analysis, food pathogen screening,complete blood count (“CBC”), hemoglobin diagnostics, infectious diseasetesting (e.g., a multi-analyte rapid diagnostic test for detectingmalaria infection), cholesterol screening, hormone testing, cardiacpulmonary, gastroenterology, urology, renal, dermatology, neurology,pediatrics, surgical, public health, and veterinary and plant pathologytesting, combinations thereof, and the like.

In addition to the foregoing, another embodiment of a lateral flowimmunoassay cassette is described. Examples of such lateral flowimmunoassay cassettes are shown at 200 in FIGS. 2A and 2B and at 300 inFIGS. 3A and 3B. In the lateral flow immunoassay cassettes 200 and 300,a test sample (i.e., a sample containing an unknown concentration of ananalyte of interest) may be run in parallel with a calibration standard(i.e., a sample containing a known concentration of the analyte ofinterest). The response to the known concentration of the analyte ofinterest in the calibration standard on the lateral flow immunoassaydevice may be used to generate a calibration curve that can be used toquantify the amount of the analyte of interest in the test sample.

Such an arrangement may provide superior results. For example, the testand calibrations strips of such cassettes may be manufacturedside-by-side under substantially equal temperature and humidityconditions. As a result, it is generally the case that the test andcalibrations strips each have the same amount on antibody immobilizedthereon and that the antibody on each will react substantially the same.Also, because the test and calibration assays are run in parallel, thetest and calibration results are generally unaffected by factors liketemperature and humidity. This is generally not the case if the test andcalibration assays are run at separate times on strips that may havebeen manufactured at different times. Likewise, because the test andcalibration assays are run in parallel, the cassettes and a readerdevice, if used, are calibrated for each assay run on each cassette,which is believed to provide more reliable quantitative results.

The lateral flow immunoassay cassette 200 illustrated in FIGS. 2A and 2Bincludes a base 214 that includes a test strip 201 a and a calibrationstrip 201 b. The test strip 201 a includes a sample application zone 202a with a sample collection pad 216 a, a conjugate pad 204 a, a testassay strip 206 a (e.g., a nitrocellulose (“NC”) membrane), and anabsorbent pad 212. Likewise, the calibration strip 201 b includes asample application zone 202 b with a sample collection pad 216 b, aconjugate pad 204 b, a calibration strip 206 b, and the absorbent pad212. Each of the test assay strip 206 a and the calibration strip 206 binclude at least one capture binding moiety 208 a and 208 b (e.g., anantibody, a nucleic acid, or the like) that can specifically interactwith and capture the analyte of interest for detection. In oneembodiment, the sample pad 212 may include flow indicator lines 210 aand 210 b (e.g., a water soluble dye) that indicate whether or notsample has successfully diffused through the test strip 201 a and thecalibration strip 201 b.

In the illustrated embodiment, the test 201 a and calibration strips 201b are run in opposite directions (i.e., both the test sample andcalibration standard flow toward absorbent pad at the center of thecassette). In other embodiments, the test and calibration strips may bearranged such that the test sample and calibration standard flowparallel to one another. Such an embodiment may, for example, include adivider arranged between the test assay strip and the calibration assaystrip.

The lateral flow immunoassay cassette 300 illustrated in FIGS. 3A and 3Bis similar to the cassette 200 of FIGS. 2A and 2B. The lateral flowimmunoassay cassette 300 includes a base 314 that includes a test strip301 a and a calibration strip 301 b. The test strip 301 a includes asample application zone 302 a with a sample collection pad 316, aconjugate pad 304 a, a test assay strip 306 a (e.g., a nitrocellulose(“NC”) membrane), and an absorbent pad 312. In addition, the test strip301 a includes includes a sachet 320 (e.g., a blister pack) of bufferthat can be used to chase (i.e., wash) a test sample through theconjugate pad 304 a and the assay strip 306 a toward the absorbent pad312.

In contrast to the cassette 200 of FIGS. 2A and 2B, the cassette 300omits a calibration standard application zone and instead includes astandard solution sachet 318 that contains a known volume of a solutionthat contains a known amount of at least one analyte of interest. Whenthe a standard solution sachet 318 is pierced at the time of use, thesolution wicks through the conjugate pad 304 b and the calibration strip306 b toward the absorbent pad 312. Each of the test assay strip 306 aand the calibration strip 306 b include at least one capture bindingmoiety 308 a and 308 b (e.g., an antibody, a nucleic acid, or the like)that can specifically interact with and capture the analyte of interestfor detection. The characteristics of the standard solution sachet 318can be used to test for quantitative delivery of the calibrationstandard onto the calibration strip 306 b and to test the response ofthe capture binding moiety 308 b to the analyte of interest. In oneembodiment, the sample pad 312 may include flow indicator lines 310 aand 310 b (e.g., a water soluble dye) that indicate whether or notsample has successfully diffused through the test strip 301 a and thecalibration strip 301 b.

In one embodiment, the sample pad 216 a, 216 b, or 316 may be configuredto absorb and dispense a predetermined amount of a fluid from the fluidthat is applied thereto. That is, the sample pad 216 a, 216 b, or 316may be fabricated from an absorbent-type material that may saturatedwith fluid and then when, for example, the sample pad 216 a, 216 b, or316 is compresses or squeezed, the sample pad 216 a, 216 b, or 316 candispense a predetermined amount of a fluid therefrom. In one embodiment,the sample pad 216 a, 216 b, or 316 may be made of cellulose, glassfiber or other material where the fluid sample is applied to the lateralflow device and, if necessary modifies it to improve the results of theassay. This might be by modifying pH, filtering out solid components,separating whole blood constituents, adsorbing out unwanted antibodiesor some other test specific variable.

For some applications, the sample pad 216 a, 216 b, or 316 may bepretreated by dipping it into a specific buffer containing a mix of asolution comprised of soluble proteins, surfactants/detergents, andother polymers. These may allow for a steady flow and preventnonspecific binding of sample components to the pad 216 a, 216 b, or316.

In some embodiments, the sample may be added to the sample pad 216 a,216 b, or 316 by collecting a liquid sample (e.g., blood, urine, orsaliva) and adding a selected volume of the sample to the sample pad. Inother embodiment, the sample may be added to the sample pad 216 a, 216b, or 316 by soaking the pad with a fluid sample. For example, thesample pad 216 a, 216 b, or 316 may be soaked with saliva by insertingthe sample collection pad 216 a, 216 b, or 316 end of the device 200 or300 into the mouth to collect a saliva sample.

In one embodiment, the conjugate pad 204 a, 204 b, 304 a, 304 b is madeof a non-absorbent material such as fiberglass pad, polyester, rayon ora similar material. The conjugate pad 204 a, 204 b, 304 a, 304 b istypically fabricated from a synthetic material (at least when using agold conjugate) to ensure the efficient release of its contents.

As its name implies, the assay's detection conjugate (e.g., colloidalgold) is dried down and held in place in the conjugate pad 204 a, 204 b,304 a, 304 b until a liquid test sample is applied to the sample pad.The liquid from the sample, by capillary action moves into the conjugatepad 204 a, 204 b, 304 a, 304 b, re-hydrates the dry conjugate and allowsthe mixing of the sample with the conjugate. The complex of conjugateand analyte then moves into and up the assay strip 206 a, 206 b, 306 a,306 b. Pretreatment of the conjugate pad 204 a, 204 b, 304 a, 304 bhelps to ensure the conjugate releases at the proper rate and enhancesits stability. The pretreatment is performed in the same way as with thesample pad 216 a, 216 b, or 316.

In one embodiment, the at least one capture binding moiety 208 a, 208 b,308 a, 308 b may be added to the test or calibration strips with adispenser that gently slides a soft capillary tube across the membrane.A dispenser pump releases a constant volume of the reagents down thelength of the membrane. This system is simple, easy to use, and lowcost. They can be somewhat cumbersome in large scale manufacturing andmany systems require a technician to constantly feed the nitrocellulosecards and to monitor reagent levels as well as the quality of the testand control lines.

An alternative method of applying the at least one capture bindingmoiety 208 a, 208 b, 308 a, 308 b includes a non-contact aerosol system.These sprayers dispense solutions in controlled ultrafine, ultra- smallvolume aerosols. These devices project very fine droplets of reagentonto the membrane and overlap the drops to create a continuous line.Spraying offers much more control of the reagent application, but italso adds capital expense and increases the complexity of stripmanufacturing. These devices are more appropriate in very large scalemanufacturing or when a reader with tight tolerances will be used toanalyze the lateral flow test strips.

In the foregoing, addition of one line of the at least one capturebinding moiety 208 a, 208 b, 308 a, 308 b onto each of the test orcalibration strips is discussed. However, one will appreciate that acassette 200 or 300 may include multiple test and control lines that mayeach be configured to interact with a different analyte of interest.

Referring now to FIGS. 4A and 4B, plan and side views of a diagnostictest system 240 are illustrated. In one embodiment, the diagnostic testsystem 240 may include a handheld device 250 and a testing apparatus260.

In the illustrated embodiment, the handheld device 250 is an iPhone.However, the handheld 250 device can be essentially any cell phonedevice, digital camera device, or a similar device that has an onboardcamera/image capture function, data collection and analysiscapabilities, data and results display capabilities, and, preferably,the ability to communicate with one or more remote computer or cellphonenetworks for data upload, querying a data analysis algorithm, querying adecision support algorithm, and the like. In the illustrated embodiment,the handheld device 250 includes a front-directed camera 280, aback-directed camera (not shown) that is directed into the testingapparatus, a display screen 290, and audio input and output ports 295 aand 295 b. The display screen 290 can be used for display of data andresults. In addition, the display screen 290 may include touchscreencapabilities that can be used for input of data or commands.Additionally the front-directed camera can be used for imaging QR andbar code information identifying the test to be performed and providinglot number, expiration date, and control values as well as otherparameters as needed for test identification, calibration, resultsinterpretation, and data reporting.

In one embodiment, the testing apparatus 260 is designed to be securelycoupled to the handheld device 250. For example, the testing apparatus260 may be designed to fit a specific class or brand of handhelddevices. The testing apparatus includes a cassette port 270 that isdesigned to allow an assay device, such as a lateral flow immunoassaycassette 105 (see FIG. 1), to be inserted into the testing apparatus260. Additionally, an interior portion of the testing apparatus 260 maybe painted with a flat black color so as to avoid extraneous andreflected light. In addition, the testing apparatus 260 includes anumber of internal components (e.g., i/o ports, power ports, lightsource(s), lens(es), light conducting media, etc.) that are designed totransform the handheld device 250 into a device that can be used tocollect and analyze data produced by an assay device, such as thelateral flow immunoassay cassette 105 (see FIG. 1).

While the testing apparatus 260, is shown fitted to the handheld device250, one will appreciate that they testing apparatus can be configuredas a separate unit that includes its own light source, power supply,optics, data capture capabilities, and the like. In such an embodiment,the testing apparatus may be configured to collect assay data from anassay cassette and transfer it (e.g., by a wired or wireless connection,by Bluetooth™, or the like) to the handheld device for analysis andreporting.

Referring now to FIG. 5A, FIG. 5A illustrates an exploded view of thediagnostic testing system 240 that is illustrated in FIGS. 4A and 4B. Ascan be seen in the exploded view, the testing apparatus 260 includes amain body housing 310 and an assay housing 320.

The main body housing 310 is primarily designed to mate cleanly with thehandheld device 250. For example, the main body housing 310 may beshaped such that the handheld device 250 can be slid into the main bodyhousing 310 such that the handheld device 250 clicks into or otherwisesecurely mates with the main body housing 310. The main body housing 310may also include one or more gaskets, seals, and the like that allow thehandheld device to form a secure and light-tight seal with the main bodyhousing 310. Additional features of the main body housing 310 will bediscussed below.

The assay housing 320 is fixedly coupled to the main body housing 310.In the illustrated embodiment, the assay housing 320 includes a cassetteport 270 that is configured such that a lateral flow immunoassaycassette 105 can be inserted into the assay housing 320. In addition,the assay housing 320 in the in the illustrated embodiment includes alens that is interposed between the handheld device's 250 back-directedcamera (not shown) and the lateral flow immunoassay cassette 105.Likewise, an optical fiber device or light pipe 340 that is capable oftransmitting light either to the lateral flow immunoassay cassette 105from the hand held device's 250 light source (not shown), from thelateral flow immunoassay cassette 105 to the hand held device's 250back-directed camera (not shown), or both.

While the hand held device's 250 light source (not shown) can be used toilluminate the lateral flow immunoassay cassette 105, the diagnostictesting system 240 may also include one or more additional light sourcesthat can be housed in either the assay housing 320 or the main bodyhousing 310. Suitable examples of light sources can include, but are notlimited to a camera flash, an autofocus illuminator on a camera, an LEDlight, an incandescent lamp, or a gas-discharge lamp. For example, thelight source can come from micro-LED lamps that are included in theassay housing 320. The micro-LEDs can be selected to emit certainwavelengths that are adapted for one or more assay conditions. Themicro-LEDs can be powered by drawing electrical power from the batteryof the handheld device 250. In addition, either the assay housing 320 orthe main body housing 310 may be configured such that ambient light orsunlight can be used to illuminate the lateral flow immunoassay cassette105.

In one embodiment, at least one wavelength filter may be interposedbetween the light source and the lateral-flow chromatographicimmunoassay cassette 105. For example, if the assay is a fluorescentassay, then the wavelength filter may be used to yield a specificwavelength of light from the light source to excite fluorescent emissionfrom the assay system. Likewise, certain colored dyes may yield a bettersignal when excited by selected wavelengths of light.

In one embodiment, the lens 330 (e.g., a collimating lens) may be usedfor focusing the light source on the lateral-flow chromatographicimmunoassay cassette 105. For example, the lens 330 may be used toincrease the amount of incident light impinging on the lateral-flowchromatographic immunoassay cassette 105. For instance, the purpose ofthe lens 330 may be to bring the focal point of the camera of thehandheld device 250 (which is limited to about 6 inches or more) to lessthan 2 centimeters. This allows for a smaller overall package andproduces a finer image that prevents the use of convoluting a blurrypicture using Fourier transforms in order to produce a usable data thatcan be analyzed. Furthermore, with a multi-analyte detection assay(e.g., two calibration standard lines and a test sample line), the finerimage will prevent overlap of the target lines to improve sensitivityand accuracy. In another example, a focusing apparatus may be used tofocus ambient light or sunlight on the analysis zone of the lateral-flowchromatographic immunoassay cassette 105.

In some embodiments, the assay cover 320 may include a device that canallow the angle of the lateral-flow chromatographic immunoassay cassette105 to be adjusted relative to the handheld device 250 and a lightsource (not shown). By selectively modifying these angles, the lowerdetection limit of the assay can be extended, the signal to noise ratiocan be improved, etc. In one embodiment, the device can be adjustedmanually in order to choose an angle that optimizes detection limit,signal to noise, and the like. In another embodiment, the device can becoupled to a mechanical means, such as a servo motor or a gel-dampedspring device that can allow the device to automatically sample a numberof angles while the handheld device 250 collects data from thelateral-flow chromatographic immunoassay cassette 105.

Referring now to FIG. 5B, the assay housing 320 and the cassette port270 are illustrated in greater detail. In the embodiment illustrated inFIG. 3B, the cassette port 270 of the assay housing 320 includes asealing gasket 350 disposed around the cassette port 270 that can sealthe cassette port 270 when an assay cassette 105 is inserted therein sothat ambient light does not leak into the housing 260. For example, ifambient light leaks into the housing 260, it could skew results. Inaddition, the cassette port 270 may include a spring-loaded flap (notshown) or similar means that can seal ambient light out of the housing260 even when no cassette 105 is inserted into the cassette port 270.

Referring now to FIGS. 6, 7A, and 7B, additional features of the housing260 are illustrated.

Referring to FIG. 6, an example of an indexing feature that can reliablyalign the housing 260 relative to the handheld device 250 isillustrated. In the illustrated embodiment, the indexing feature mayinclude a headphone jack 410 that is integrated into the housing body310. When the handheld device 250 is inserted into the housing body 310,the headphone jack 400 is positioned such that it can be inserted intothe headphone port 410 of the handheld device 250. It will be understoodby persons having ordinary skill in the art that headphone jack 400 isbut one example of an indexing feature and that additional indexingfeatures can be employed without departing from the spirit of thisdiscussion.

In addition to aligning the housing body 310 relative to the handhelddevice 250, the headphone jack 400 can be used to draw electrical powerfrom the handheld device 250 in order to power components (e.g., one ormore illumination devices) that are positioned in the housing 260.Likewise, the headphone jack 400 can be used for data transfer betweenthe handheld device 250 and components in the housing 260.

Referring now to FIG. 7A, a target device 500 is illustrated. The targetdevice 500 can be used to normalize/calibrate the response of at leastone of the camera or the light source of the handheld device. In oneembodiment, the target device may located on an interior surface 325 ofthe assay housing 320 in close proximity to the cassette port 270 in anarea that is can be illuminated by a light source that will be employedfor illumination of an assay cassette and viewable by a camera of ahandheld device that is going to be used to capture data from thecassette. For example, the target device may have a known color and/orcolor intensity that can give a known response for calibrating the lightsource and the camera. In addition, the target device 500 can be used toensure that the light source and the camera are directed at the properpoint when the handheld device in inserted into the housing.

Referring now to FIGS. 7A and 7B, the assay housing 320 may furtherinclude a mechanical interlocking feature 510 that is positioned andconfigured to mate with a mechanical interlocking feature 520 on theassay cassette. For example, the mechanical interlocking features 510and 520 may include tab and cut-out features that are designed to fittogether. Such mechanical interlocking features 510 and 520 may be usedto ensure that the cassette 105 is inserted in to the assay housing 320in the proper orientation. In addition, such mechanical interlockingfeatures 510 and 520 may be coupled to a disabling feature that can shutdown the device if an incompatible cassette is inserted into the housing320 or if the cassette is inserted in the wrong orientation. This can,for example, be an important safety feature because it prevents thedevice from reading the wrong portion of the cassette and giving anerroneous reading as a result.

II. Methods for Detecting at Least One Analyte of Interest in a Sample

In one embodiment, a method for detecting at least one analyte ofinterest in a sample is disclosed. The method includes providing alateral-flow chromatographic assay cassette and providing a testingdevice that is capable of interfacing with the lateral-flowchromatographic assay cassette.

In an embodiment, the lateral-flow chromatographic assay cassette mayinclude a capture ligand capable of capturing and localizing at leastone analyte of interest in a sample on an analysis surface of thelateral-flow chromatographic assay cassette, at least one reporterconfigured for interacting with at least one of the analyte of interestor the capture ligand, and at least a first calibration standard and asecond calibration standard configured to provide at least a two-pointcalibration curve. In another embodiment, a lateral-flow chromatographicassay cassette may include an absorbent test strip for analyzing ananalyte of interest in an experimental sample and an absorbentcalibration strip for running at least one calibration standardpositioned in proximity to the absorbent test strip as described ingreater detail elsewhere herein.

In one embodiment, the lateral-flow chromatographic assay cassette maybe packaged in a packaging system 600, as illustrated in FIG. 8. Thepackaging system 600 includes a sealed package (e.g., a plastic-, foil-,or paper-based package) that can be used for containing, storing, ortransporting the lateral-flow chromatographic assay cassette 610 in aclean and preferably sterile environment. A QR code decal or stickerwith relevant cassette information could be applied or printed to theoutside of each foil pouch or canister.

In addition, the packaging system 600 includes a tracking code 630. Inthe illustrated embodiment, the tracking code 630 is a QR code, which isa two-dimensional bar code. Two-dimensional bar codes, like QR codes,can be used to store far more information that can be stored in aconventional bar code. For example, a QR code can be used to store up˜4300 alphanumeric characters (i.e., 0-9, A-Z, space, $, %, *, +,−, .,/, :, etc.). In one embodiment, the tracking code 630 can be read by thediagnostic testing system prior to initiating a test. The tracking codemay be used to store information that is relevant to the test in aformat that can be read by the device. For example, the tracking code630 can be used for recording and then transmitting to the test systemthe values for the calibration standards used on the lateral-flowchromatographic assay cassette 610, manufacturer, date of manufacture,lot number for the lateral-flow chromatographic assay cassette 610,manufacturer, date of manufacture, and sample/results tracking.

The testing device may include a testing apparatus that is configured tocouple the lateral-flow chromatographic assay cassette to the handhelddevice in proximity to a light source, the light source being capable oftransmitting at least one wavelength of light configured to yield adetectable signal from the reporter(s) (e.g., at least one reporterconfigured for interacting with at least one of the analyte of interestin a test sample and/or a calibration sample, the first calibrationstandard, and the second calibration standard), and a detector ispositioned to capture the detectable signal from the reporter(s).

The method may further include applying a liquid sample that includes atleast one analyte of interest to the lateral-flow chromatographic assaycassette. In some embodiments, applying a liquid sample to the cassettemay include applying separate test and calibration samples to separatetest and calibration strips. The method may further include insertingthe lateral-flow chromatographic assay cassette into the testing device,illuminating the lateral-flow chromatographic assay cassette with thelight source of the testing device in order to yield a detectable signalfrom the reporter(s), and querying an interpretive algorithm for (i)calculating the calibration curve and then (ii) converting thedetectable signal from the first reporter to a numerical value relatedto the presence or amount of the at least one analyte present in asample.

In one embodiment, the calibration curve may be calculated based onvalues from interaction of a first calibration standard and a secondcalibration standard with calibration standard lines on the cassette.See, e.g., calibration standards lines 150 a and 150 b of FIG. 1. Inanother embodiment, the calibration curve may be calculated based on (1)observing a blank region of the absorbent calibration strip, and (2)generating a two point calibration curve that includes a value for theinteraction of the analyte of interest from the liquid calibrationstandard with the ligand immobilized on the absorbent calibration stripand a value for the blank region of the absorbent calibration strip. Anexample of the blank region on a calibration strip 206 b and 306 b isillustrated at 222 and 322 in FIGS. 2A and 3A. Because the calibrationstrip may not be pure white, the strip may produce a background signalthat needs to be subtracted to get a true value for the signal from thetest and calibration lines. Moreover, instead of assuming a zero value,observing the background signal in the blank region allows thecalculation of a true two-point calibration curve, which is moreaccurate.

In one embodiment, the method may further include providing means fordispensing a known amount of liquid from a sample pad of the assaycassette. Such means may include, without limitation, rollers, presses,rollers or presses that include a stop that determines how much liquidcan be squeezed from the samples pad, spring loaded devices thatautomatically press down on the sample pad to dispense a predeterminedamount of liquid, and the like. In one embodiment, the testing devicemay include means for dispensing a known amount of liquid from thesample pad. For example, the testing device may include a port forinserting the lateral-flow chromatographic assay cassette into thetesting device. Such a port may, for example, include a roller or asimilar means that rolls over the sample pad and dispenses a selectedamount of liquid therefrom when the cassette is inserted into thetesting device.

In one embodiment, a single immunoassay device may contain multipletypes of different capture moieties (e.g., antibodies) each conjugatedwith different dyes (e.g., quantum dots) and/or multiple capture bandseach immobilized with different capture moieties. A single light source(e.g., an ultraviolet light) illuminates all dyes (e.g., quantum dots)simultaneously, and the detector device (e.g., a digital camera)captures the emitted signals from multiple bands simultaneously.

In one embodiment, analytes of interest assayed on the lateral flowimmunoassay cassettes described herein may be detected and quantified byelastic light scattering. The amount of light scattered from a selectedregion of a lateral flow immunoassay cassette (e.g., a capture band) ishighly sensitive to the amount of material in a region illuminated by anincident light. In general, elastic light scattering, coupled with angleoptimization, may be as much as 100 times more sensitive than comparablereflectance or fluorescence analysis. Other excitation/detection methodsmay include surface plasmon detection; Rayleigh scattering, reflectance,diffuse scattering, electrochemical detection, conductivity,fluorescence, magnetic, enzymatic, transmission, absorption, acousticdetection, any other method which is based upon Beer's law, kineticanalysis (e.g., change in signal strength over time), and the like.

In one embodiment, a light source may be positioned at a certain angleto the lateral flow assay cassette and the detector (e.g., a detectionfiber or a cell phone camera) or fiber (eventually the cellphone cameraCCD). In one embodiment, the reporter(s) may be queried by taking areading from each reporter and calculating the intensity of thescattered light. Signal intensity (i.e., the amount of scattered lightthat is detected) decreases as the concentration of the analyte ofinterest increases.

In an embodiment that includes a cell phone camera or the like, thecamera's CCD will take an image. In one embodiment, the image may betaken with a red distance filter. In the image, the calibration standardlines and the test lines will be present. The digital image will thenundergo digital image processing with a selected digital processingalgorithm to produce a representative image of the color bands for thecalibration standard lines and test simultaneously. For example, adigital processing algorithm may (1) identify the areas of interest(e.g., the test line and the at least two calibration standard lines) inthe image taken of the lateral flow immunoassay cassette, (2) calculatean RGB value for each pixel in the image, (3) convert RGB format to xyzformat, (4) convert xyz format to Lab color format, (5) assign anumerical value to each of the areas of interest (e.g., the test lineand the at least two calibration standard lines), (6) calculate acalibration curve based on the numerical values obtained from the firstand second calibration standard lines values, and (7) convert thenumerical value for the test line into a concentration value for theanalyte of interest in the sample.

In addition, internal controls, such as but not limited to, a controlline (e.g., a fluorescent marker) to potentially eliminate or reducevariations in the final signal from manufacturing tolerances of thelateral flow assay cassette may be used to increase the robustness andreliability of the analysis. Additionally, analysis of the white portionof the lateral flow assay cassette may be used as an additional negativecontrol to further improve reproducibility.

The digital processing algorithm is able to convert the numerical valuefor the test line into a concentration value because the at least twocalibration standard lines are selected to provide numerical values thatare proportional to non-zero concentration amounts for the analyte ofinterest. This relationship is clarified by reference to FIG. 9, whichshows a graph 700 with Lab value on the Y-axis and concentration on theX-axis. The first and second calibration standards have a known responsethat relates to known and, preferably, non-zero concentration values forthe analyte of interest. Lab values for each of the first and secondcalibration standards 730 and 740 can be related to a concentration foreach 750 and 760 by a simple relationship. By relating observed Labcolor values to concentration values 750 and 760, a calibration curve770 can be generated that can be used to calculate the concentration 790of the analyte of interest in the sample based on the observed Lab color780. One will of course appreciate that the calibration curve 770 canalso be described by a mathematical formula and that the analysisalgorithm may not actually generate a calibration curve, per se.

In one embodiment, the method may further include mixing the liquidsample with a dye conjugate prior to applying the sample to thelateral-flow chromatographic immunoassay cassette. In one embodiment,the dye conjugate is configured to interact with at least one of theanalyte of interest or the ligand to provide a visual readout related tothe presence or concentration of the analyte of interest in the sample.In one embodiment, the sample includes at least one control substanceand at least one analyte of interest.

In one embodiment, the observation of the interaction of the at leastone analyte of interest with the at least one ligand immobilized on thelateral-flow chromatographic immunoassay cassette may be timed byobserving the appearance of at least one control substance. For example,a thyroid stimulating hormone (“TSH”) assay may be read ˜10 minutesafter a diluent is applied. By monitoring the position of the wave frontor the appearance of the control line, it may be possible to eliminatethe need to manually time the test. Likewise, by observing the timing ofthe appearance of a control, the most favorable time for reading theassay can be identified. These could include monitoring the movement ofthe mobile phase, monitoring the movement of the control substance,timing the movement of the mobile phase, taking sequential images of thetest result, detecting when buffer is added, detecting when liquid hastraveled the length of the membrane, and combinations thereof.

In addition, testing device may include or may be configured to accessan interpretive algorithm stored in a computer readable format andelectronically coupled to the handheld device, wherein the interpretivealgorithm is configured to (i) calculate a calibration curve based onthe first calibration standard and the second calibration standard andthen (ii) convert the detectable signal from the first reporter to anumerical value related to the presence or amount of the at least oneanalyte present in a sample. The interpretive algorithm may be includedin an on-board computing system of the handheld device or theinterpretive algorithm may be stored remotely in a computer storagemedium that is accessible by the handheld device.

In one embodiment, the interpretive algorithm queried in the abovedescribed method may include one or more computer storage media havingstored thereon computer executable instructions that, when executed byone or more processors of the detector device, implement a method forinterpreting the numerical value related to the presence or amount ofthe at least one analyte present in the sample. In one embodiment, thecomputer implemented method may include (1) receiving a user initiatedrequest to convert the visual signal readout of the immunoassayapparatus to a numerical value, (2) in response to the request, an actof identifying at least one visual signal readout of the immunoassayapparatus, (3) capturing at least one digital signal from the at leastone visual signal readout of the immunoassay apparatus, (4) convertingthe at least digital signal to at least one numerical, and (5) using theat least one numerical value to determine an amount or concentration ofat least one analyte present in the sample. This numerical value canthen be displayed on a screen located on the detector device and/orstored, interpreted, or sent to a database.

In one embodiment, the computer implemented method may further includeat least one of: (1) communicating with an electronic medical recordssystem via a wireless communication channel, (2) uploading the amount orconcentration of the at least one analyte present in the sample to theelectronic medical records system, or (3) querying a decision supportalgorithm, wherein the decision support algorithm uses the at least onenumerical value to support a diagnosis of at least one condition in asubject and to suggest a course of treatment.

FIG. 10 schematically illustrates the decisions that may be made oractions that may be taken in an example decision support algorithm for athyroid stimulating hormone (TSH) test. At the first branch point, ifTSH is normal then no action is taken. If TSH is low, a clinician willbe directed to check free thyroxine (T4). If free T4 is normal, thealgorithm directs that the test should be repeated in 3-6 months; iffree T4 is high or low, the algorithm directs that the patient should bereferred to a specialist. If at the first branch point TSH is high, theclinician will be directed to check free T4. If free T4 is normal, thealgorithm directs that the test should be repeated in 3-6 months; iffree T4 is high, the patient should be referred to a specialist; and iffree T4 is low, the algorithm directs that the patient should receive atreatment for hypothyroidism.

Embodiments of the present disclosure may comprise or utilize specialpurpose or general-purpose computing devices that include computerhardware, such as, for example, one or more processors and systemmemory, as discussed in greater detail below. Embodiments within thescope of the present invention also include physical and othercomputer-readable and recordable type media for carrying or storingcomputer-executable instructions and/or data structures. Suchcomputer-readable recordable media can be any available media that canbe accessed by a general purpose or special purpose computer system.Computer-readable media that store computer-executable instructionsaccording to the invention are recordable-type storage media or otherphysical computer storage media (devices) that are distinguished frommere transitory carrier waves.

Computer-readable media that carry computer-executable instructions aretransmission media. Thus, by way of example, and not limitation,embodiments of the invention can comprise at least two distinctlydifferent kinds of computer-readable recordable media: computer storagemedia (devices) and transmission media.

Computer storage media (devices) includes RAM, ROM, EEPROM, CD-ROM orother optical disk storage, magnetic disk storage or other magneticstorage devices, or any other medium which can be used to store desiredprogram code means in the form of computer-executable instructions ordata structures and which can be accessed by a general purpose orspecial purpose computer and which are recorded on one or morerecordable type medium (device).

A “network” is defined as one or more data links or communicationchannels that enable the transport of electronic data between computersystems and/or modules and/or other electronic devices. When informationis transferred or provided over a network or another communicationsconnection or channel (either hardwired, wireless, or a combination ofhardwired or wireless) to a computer, the computer properly views theconnection as a transmission medium. Transmissions media can include anetwork and/or data links which can be used to carry or desired programcode means in the form of computer-executable instructions or datastructures and which can be accessed by a general purpose or specialpurpose computer. Combinations of the above should also be includedwithin the scope of computer-readable media.

Further, upon reaching various computer system components, program codemeans in the form of computer-executable instructions or data structurescan be transferred automatically from transmission media to computerstorage media (devices) (or vice versa). For example,computer-executable instructions or data structures received over anetwork or data link can be buffered in RAM within a network interfacemodule (e.g., a “NIC”), and then eventually transferred to computersystem RAM and/or to less volatile computer storage media (devices) at acomputer system. Thus, it should be understood that computer storagemedia (devices) can be included in computer system components that also(or even primarily) utilize transmission media.

Computer-executable instructions comprise, for example, instructions anddata which, when executed at a processor, cause a general purposecomputer, special purpose computer, or special purpose processing deviceto perform a certain function or group of functions. The computerexecutable instructions may be, for example, binaries, intermediateformat instructions such as assembly language, or even source code.Although the subject matter has been described in language specific tostructural features and/or methodological acts, it is to be understoodthat the subject matter defined in the appended claims is notnecessarily limited to the described features or acts described herein.Rather, the described features and acts are disclosed as example formsof implementing the claims.

Those skilled in the art will appreciate that the invention may bepracticed in network computing environments with many types of computersystem configurations, including, personal computers, desktop computers,laptop/notebook computers, message processors, hand-held devices,multi-processor systems, microprocessor-based or programmable consumerelectronics, network PCs, minicomputers, mainframe computers, tablets,mobile telephones, PDAs, pagers, routers, switches, and the like. Theinvention may also be practiced in distributed system environments wherelocal and remote computer systems, which are linked (either by hardwireddata links, wireless data links, or by a combination of hardwired andwireless data links) through a network, both perform tasks. In adistributed system environment, program modules may be located in bothlocal and remote memory storage devices.

In particular, one or more embodiments of the invention may be practicedwith mobile consumer computing devices. Mobile consumer computingdevices or more simply, mobile consumer devices, can be any of a broadrange of computing devices designed or optimized for portability and forpersonal use. Mobile consumer devices can take a variety of forms,ranging from more traditional notebook and netbook computers to anemerging and rapidly growing market of handheld devices, including smartphones (e.g., the APPLE IPHONE, ANDROID phones, WINDOWS phones, SYMBIANphones), tablet computers (e.g., the APPLE IPAD, ANDROID tablets),gaming devices (e.g., NINTENDO or PLAYSTATION portable gaming devices,the APPLE IPOD), multimedia devices (e.g., the APPLE IPOD), andcombinations thereof. Many of these devices can enable richuser-interactivity by including combinations of output, input, and othersensory devices, such as touch- or pressure-sensitive displays (usingcapacitive or resistive technologies, for example), still and videocameras, Global Positioning System (GPS) receivers, magnetic compasses,gyroscopes, accelerometers, light sensors, proximity sensors,microphones, speakers, etc. These devices can also comprise a variety ofcommunications devices, such as combinations of cellular modems (e.g.,Global System for Mobile Communications (GSM), Code division multipleaccess (CDMA)), Wireless Fidelity (Wi-Fi) radios, Bluetooth radios, NearField Communication (NFC) devices, etc. Many mobile consumer devices areexpandable, such that a user can add new hardware and functionality notpresent during manufacture of the device. It will be appreciated that asthe market for mobile consumer devices expands and develops, thefunctionality of these devices will also expand to utilize new andimproved user-interaction devices and communications devices. Theembodiments described herein are expansive and can also utilize anyfuture developments in the field of mobile consumer devices.

EXAMPLE

The following Example describes an example of a test device thatincludes an iPhone and a test device coupled to the iPhone. The testdevice includes a slot for inserting a lateral flow assay cassette intothe test device for reading and analysis by the iPhone.

There are a couple of challenges to imaging the measurement cassette.The first is to fill the iPhone's camera frame with as much of thedetection strip as possible. This suggests a short distance between thecamera and cassette. The second challenge is to evenly illuminate thedetection strip to make image processing easier. This requirementsuggests a longer distance.

Generally, even illumination is the more challenging requirement. In oneembodiment, a light pipe or a similar device may be interposed betweenthe illumination source (e.g., the iPhone's flash or another lightsource that is included in the test device). Light pipes arecommercially available in various configurations, such as, but notlimited to, cylinders and rectangles. The rectangle shape has beentested and been found to work better than the cylindrical configuration.The physical dimensions of the rectangular light pipe are in thefollowing document online http://www.lumex.com/specs/LPB-R0112051S.pdf,the entirety of which is incorporated herein by reference.

As described above with respect to the Figures, the test device mayinclude an accessory lens that is disposed between the camera's lens andthe lateral flow assay cassette. The lens currently being tested has a20 mm focal length and 6 mm diameter. This lens was ordered fromThorlabs.com with physical dimensions selectable in several formatsfrom: http://www.thorlabs.us/thorProduct.cfm?partNumber=LA1700-A, thePDF version is: http://www.thorlabs.us/Thorcat/4400/4414-E0W.pdf, theentireties of which are incorporated herein by reference. A 30 mm focallength should be a good value for filling the iPhone camera's frame andachieving even illumination of the detection strip. A focal length of 60mm is also an interesting choice since the iPhone may not need a secondlens. However, this may potentially limit sensitivity in the finalmeasurement.

One will of course appreciate that either the light pipe or the lens mayinclude one or more light filters that allow selective illumination ofthe detection strip and/or detection of selection wavelengths of lightfrom the detections strip. Likewise, the test device may include one ormore light sources that emit selected wavelengths of for illumination ofthe detection strip. Analysis of images or a detection strip configuredfor detection of TSH with colloidal gold with a properly configuredlight pipe show dips in reflectivity in all three color channels (red,blue, green). With a proper exposure, there is a greatest difference inthe green channel, corresponding to the 580 nm peak in the reflectancespectrum. The green channel shows a difference for both controls and themeasured sample. This suggests that it may be best to illuminate with aselected wavelength of light that gives the best signal-to-noise ratiofor detection of signal from colloidal gold when observing in thevicinity of 580 nm.

In this Example, there are two large changes relative to the deviceshown and discussed with respect to the Figures. Both of these changesrelate to the orientation of the cassette. In this version the cassetteis flat relative to the iPhone body and the long axis of the cassettebeing aligned with the long axis of the iPhone body. The image sensor inthe iPhone is asymmetrical with the long axis of the image sensor beingaligned with the long axis of the phone body. Orienting the long axis ofthe detection strip with the long axis of the phone orients thedetection strip with the axis of the image sensor that contains the mostpixels. The distance between the camera body and the cassette should bethe focal length of the lens, in the present configuration 30 mm.

The center of the measurement part of the cassette where the sampleshould be on axis with the center of the camera lens. The center of thelight pipe should be in the center of the LED lamp and oriented with itslong dimension along the long dimension of the camera. The cut out forthe lens and the cut out for the light pipe will leave a fairly thinwall between the two cut outs. Placing a thin wall between the lightpipe and the lens prevent the lens from being affected by light comingdirectly from the illumination source. In addition, it has been observedthat the color of the body of the smartphone can affect illumination andthe results obtained from an assay. For instance, it was observed thatlight from a white iPhone flash diffuses through the plastic case morethan the light from a black iPhone flash. This confounding factor can,for example, be addressed by an algorithm correction or by placing agasket or physical barrier around the flash to limit and control lightdiffusion.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed is:
 1. A diagnostic test system for performing a pointof care diagnostic test for detecting and quantifying at least oneanalyte in a biological sample, comprising: a lateral-flowchromatographic assay cassette that includes: a capture ligand capableof capturing and localizing at least one analyte of interest in a sampleon an analysis surface of the lateral-flow chromatographic assaycassette, at least one reporter for visualizing the interaction of theanalyte of interest and the capture ligand, and an assembly forproviding an at least a two-point calibration curve for quantificationof the at least one analyte of interest; a testing device comprising ahandheld computing device selected from the group consisting of ahandheld digital camera device, a cellular phone, a smart phone, and atablet computer; a testing apparatus configured to be coupled to thehandheld computing device and configured to position the lateral-flowchromatographic assay cassette in proximity to a detector and a lightsource to control focal length from the detector to the lateral-flowchromatographic assay cassette and to control illumination of thelateral-flow chromatographic assay cassette by the light source, whereinthe testing apparatus comprises an assay cover including a manual orelectromechanical device configured to allow the angle of the lateralflow assay cassette to be adjusted relative to the light source tosample a number of angles while the handheld computing device collectsdata to determine the optimum angle for detection; the light sourcebeing capable of transmitting at least one wavelength of lightconfigured to yield a detectable signal from the at least one reporter;a detector positioned to capture the detectable signal from the at leastone reporter; a collimating lens interposed between the detector, andthe analysis surface of the lateral-flow chromatographic assay cassette;and an interpretive algorithm stored in a computer readable format andelectronically coupled to the testing device, wherein the interpretivealgorithm is configured to (i) calculate a calibration curve and then(ii) convert the detectable signal from the at least one reporter to anumerical value related to the presence or amount of the at least oneanalyte present in a sample, wherein the assembly for providing an atleast a two-point calibration curve includes at least one of: alateral-flow chromatographic assay cassette that includes at least afirst calibration standard and a second calibration standard configuredto provide at least a two-point calibration curve, or a lateral-flowchromatographic assay cassette that includes a test strip and a separatecalibration strip cassette, wherein the calibration strip includes acapture ligand configured to capture a known amount of the analyte ofinterest.
 2. The diagnostic test system of claim 1, wherein the testingapparatus is physically coupled to the testing device.
 3. The diagnostictest system of claim 2, wherein the testing apparatus includes at leastone indexing feature configured to align the testing apparatus with thetesting device.
 4. The diagnostic test system of claim 2, wherein thetesting apparatus includes one or more seals to create a light-tightenvironment between the testing device and the testing apparatus andbetween the testing apparatus and the lateral-flow chromatographic assaycassette.
 5. The diagnostic test system of claim 1, wherein the lightsource is at least one of a camera flash, an autofocus illuminator,ambient light, sunlight, an LED light, an incandescent lamp, or agas-discharge lamp.
 6. The diagnostic test system of claim 5, wherein atleast one focusing lens is interposed between the light source, thedetector, and the analysis surface of the lateral-flow chromatographicassay cassette.
 7. The diagnostic test system of claim 5, wherein atleast one wavelength filter is interposed between the light source andthe analysis surface of the lateral-flow chromatographic assay cassette.8. The diagnostic test system of claim 5, wherein at least one lightconducting fiber is interposed between the light source and the analysissurface of the lateral-flow chromatographic assay cassette.
 9. Thediagnostic test system of claim 1, wherein the at least one reporterincludes at least one of colored beads, colloidal gold, colloidalsilver, dyes, fluorescent dyes, an electrochemical detector, aconductivity detector, or quantum dots.
 10. The diagnostic test systemof claim 1, wherein the detectable signal includes at least one ofemission, reflectance, diffuse scattering, elastic light scattering,transmission, fluorescence, surface plasmon detection, or Rayleighscattering.
 11. The diagnostic test system of claim 1, the lateral-flowchromatographic assay cassette further including a tracking featurereadable by the at least one of the testing device or the testingapparatus, wherein the tracking feature provides values calculating thecalibration curve.
 12. The diagnostic test system of claim 1, thelateral-flow chromatographic assay cassette further including a firstmechanical interlock feature configured to interlock with acorresponding second mechanical interlock feature on the testingapparatus.
 13. The diagnostic test system of claim 12, wherein the firstand second mechanical interlock features are configured to align thelateral-flow chromatographic assay cassette relative to the testingapparatus, the light source, and the detector.
 14. The diagnostic testsystem of claim 12, wherein the first and second mechanical interlockfeatures are configured to disable the diagnostic test system if thefirst and second mechanical interlock features do not align when thelateral-flow chromatographic assay cassette is inserted into the testingapparatus.
 15. The diagnostic test system of claim 1, the testingapparatus further including a target device capable of being illuminatedby the light source and viewable by the detector, wherein the targetdevice is configured for normalizing and/or calibrating the light sourceand the detector.
 16. The diagnostic test system of claim 1, wherein thetesting apparatus is physically separate from the testing device, andwherein the testing device communicates with the testing apparatus viaat least one of a wired connection or a wireless communicationapparatus.